RNA Biology and Molecular Physiology
AtGRP7 Affects Alternative Splicing
Alternative splicing (AS) has an enormous impact on the transcriptome and proteome in the cell. This regulatory process can give rise to different protein isoforms with variable domain composition. AS also impacts transcript levels, e.g. by generating transcripts with premature termination codons that are degraded via the nonsense-mediated decay pathway.
As AtGRP7 negatively autoregulates its own alternative splicing, we hypothesized that it might have a broader impact on AS.
In collaboration with Dr. Craig Simpson and Prof. Dr. John Brown (The James Hutton Institute, University of Dundee) we globally compared alternative splicing events in At GRP7-ox plants and wt plants using their RT-PCR based alternative splicing panel. Approx. 20 % of the investigated alternative splicing events are significantly changed upon overexpression of At GRP7.
Strikingly, several splicing events change in opposite direction in At GRP7-ox plants and atgrp7 mutant plants, respectively, suggesting that these transcripts may be direct At GRP7 targets. This conjecture was further supported by the observation that mutation of one arginine ( At GRP7-R49Q) that reduced the RNA-binding affinity abrogated the effect. Indeed, making use of our newly established RIP protocol we show that candidate AS targets are directly bound by At GRP7-GFP in vivo but not by At GRP7- R49Q-GFP.
